Iscom or iscom-matrix comprising hydrophobic receptor molecules for antigenic substances

ABSTRACT

Lipid-containing particles such as iscoms, iscom-matrix or vesicles such as micelles or liposomes that comprise one or more hydrophobic receptor molecules for targeting and antigenic substances, which receptor molecules have been integrated in the particle and are chosen from lipid-containing receptors or receptors that are hydrophobic, which receptor molecules bind to the antigenic substances.

The invention involves lipid-containing particles, chosen from iscomsand iscom matrices, which contain one (several) hydrophobic receptorcomponent(s) which bind to antigens from microorganisms such asbacteria, virus, or parts thereof, i e the receptor-binding parts, suchas toxins or surface proteins.

Furthermore, the invention involves procedures for producing suchlipid-containing particles and the human medical, veterinary medical, orother pharmacological preventive and curative use, such asimmunotherapeutic, of these particles. The invention involves inparticular iscoms and iscom matrices whose surfaces have been preparedwith bacterial toxin fragments such as the cholera toxin's B subunit(CTB).

THE BACKGROUND OF THE INVENTION

Lipid-containing structures in the form of micelles, liposomes and othervisicles, iscoms (immune-stimulating complex/particles), iscom matrices,etc. have been reported as effective carriers of pharmacologicallyand/or immunologically active substances or molecule complexes. See forexample WO-A1-90/03184 (Morein et. al., Clin. Immunother. Review, 1995;Kersten et. al., Iscom--Liposome Review, 1995). In many cases,immunization of laboratory animals with such lipid-containingstructures, in which various antigens have been incorporated, have beenshown to give rise to an increased immune response to the referredantigens as compared to the immune response obtained after immunizationusing a corresponding antigen in a free form.

Iscom and iscom matrices are documented as effective carriers ofantigens and adjuvant molecules to enhance the immunogenicity of smalland large molecules (antigens), i e to make them strongly immunogenicboth when they are applied parenterally and locally, (topically) onmucous surfaces. The iscom has unique properties being effective aftermucosal intranasal adminstration. It is well-documented (Morein et. al.,Clin. Immunother. 3, 1995, 461-475) that both iscoms with incorporatedantigens (usually protein) and iscoms as carriers, for example smallantigens such as oligopeptides or as exemplified by biotin, effectivelyevoke immune response to these large or small molecules.

Iscom matrices (and iscom) have well-documented, built-in, adjuvantactivity that potentiates antibody-mediated as well as cell-mediatedimmune responses to the co-administered antigens. Iscom evokescell-mediated immune response under both Class I and Class IIrestriction.

Cholera is the most serious of all the diarrhea diseases and is causedby the Vibrio cholera bacteria in group 1. These bacteria colonize inthe small intenstine of human beings and secrete an exotoxin proteinknown as the cholera toxin. This toxin binds to and is absorbed by cellsin the mucous membranes and causes an intensive secretion ofelectrolytes and water from the cells, which leads to the grave cases ofdiarrhea, dehydration, and metabolic acidosis which characterizecholera.

Similar diseases can be caused by so-called "enterotoxic" (ET)cholibacteria, but the symptoms are usually milder. Such bacteria oftencause diarrhea in young individuals among humans and practically allkinds of animals, including pigs and cattle. These diarrheas, which cangive rise to great economic losses for the livestock industry, arecaused partly by a heat-labile toxin (LT) similar to the cholera toxin(CT). These toxins are so similar that they bind to the same receptors.

The structures of CT and LT are well defined in regards to structure andfunction. They are oligomeric proteins consisting of one part that bindsto the cholera toxin receptor, namely the B part, which in turn consistsof five subunits which each have an approximate mole weight of 11,600and form a pentamer ring. The A subunit is a proteolytic splitpolypeptide with a molecular weight of approximately 28,000, consistingof two disulfid-conjugated fragments. The larger A1 fragment containstoxin-enzyme activity, while the smaller A2 fragment joins the A1fragment with the B5 ring. CT binds with high affinity to a class ofreceptors that exist on the surface of the so-called brush-bordermembranes in the small intestine, as well as to the plasma membrane ofmost mammalian cells. The GM1 gangliosid constitute the receptor for CT(Holmgren et. al., Infect. Immun. 38, 424-433). LT also binds to GM1.

CT and LT, respectively, are both important components in the subunitvaccines that are intended to evoke protection gains cholera andenterotoxic cholibacteria. In the case of intestinal infections, it isof special interest to evoke local protection exerted by, among otherthings, secretory IgA in the intestinal membrane. CT and LT are bothconsidered well suited as targeting molecules in adjuvant formulationsfor vaccines intended for adminstration in the intestinal andrespiratory tracts (Morein. Lovgren and Cox, 1966), with, among otherthings, having the capacity to induce a secretory IgA response that isan important component in the protection. The B subunit of CT and LThave attracted a good deal of interest as carrier molecules and even asuniversal vector systems for oral vaccines (Mucosal Handbook Immunology,eds Ogra, P. L., Lamm Me, Mc Ghee Jr., Mestechy J., Strober W.Bienenstock J., 1994). The interest has increased even more because ithas been shown that the conjugate between CTB and other antigens notonly give rise to immune response in the local intestinal mucosalmembranes, but also to a limited extent in other remote mucousmembranes, such as the salivary glands, the lungs, the genital tract,and in the blood (Handbook mucosal, 1994). The problem with CTB and LTBis that they have a low (inate)-capacity to potentiate their own strong,protective immune response against the cholera toxin or against LT, oragainst the antigen that they are modified to be a carrier for. Theythus have a low adjuvant activity in relation to the immunomodulatoryand immunopotentiating effect (Morein, Lovgren and Cox, 1966).

CTB and LTB are used experimentally as carriers of antigen with thepurpose of evoking, through local application (orally), local immuneresponse in the mucous membranes of the digestive tract as well as inother mucous membranes through gut-associated lymphatic traffic (GALT)or through direct application on other mucous membranes such as therespiratory tract. CTB and LTB have targeting capacity, which means thatthey are considered to steer and localize both themselves and theantigens they may carry to the lymphatic system in the intestinal tract,which means to M-cells in Payer's patches, to lamina propria (LP), andto the lymphatic system in the intestines and in other mucous membranesthrough GALT or through direct application on these mucous membranes,for example in the respiratory tract.

The following unsolved difficulties exist regarding using CTB and LTBfor local immunization:

1. CTB and LTB have by their own relatively low immunogenicity and a lowimmunoenhancing capacity, requiring a need to be potentiated with anadjuvant component to obtain optimal effect. In other words, thisinvolves both their own immunogenicity and their immunoenhancing effectto the antigens that they may have carried with them. Their value asadjuvants is limited to "targeting", while supplementary adjuvantactivities in the form of immunomodulatory and immunoenhancingcapacities are required in order to attain optimal immunogenicity.

2. There are limitations to conjugating antigens to CTB and LTB withparticularly high physical or economic yield, since only a limitednumber of amino groups and/or carboxy groups can be activated withoutseriously reducing their values as antigen or as carriers in mucousmembranes, and target themselves and the accompanying antigens to thelymphatic organs and cells to evoke immune response. Even if asufficient number of coupling groups are available on a carriermolecule, it is well known that it is difficult to attain the desiredeconomic yield from such constructions because of the insufficientyield. For example, often no more than 15-20% of the available antigensare coupled in reaction to the carrier molecule.

3. CTB and LTB have a limited space for chemically coupling of largermolecules, because they can block functional epitopes that are necessaryfor targeting the complexes to the lymphatic organs and cells.

SUMMARY OF THE INVENTION

It has now been demonstrated that through the use of lipid-containingparticles, chosen from iscoms and iscom matrices or micelles or vesicleslike liposomes that contain one or more hydrophobic receptor moleculesfor antigen substances or targeting molecules, a contribution is madetoward a new, general system for binding molecules. These receptors canbe lipid-containing receptors or receptors that are hydrophobicproteins. Through this new, general system, a greater proportion ofantigens or other substances are bound to the particle, with a yieldthat begins to approach 100%, which is economically advantageous, butabove all the new system makes it possible, without competition fromearlier systems (that do not use the receptor) (EP 0 109 924 B1, EP 0180 546 B1, EP 0 242 380 B1), to bind in antigen and targeting moleculesor molecule complexes. Consequently, the immune response is moreefficiently induced by the antigens that are bound to the receptor.Above all, it becomes possible to bind antigen to the receptor togetherwith the other antigens that are incorporated without using thereceptor. With this invention it is easier to bind both targetingmolecules which can, for example, penetrate mucous membranes, andpassenger antigens which cannot be absorbed by mucous membranes (see theSwedish patent application 9600647-3). A special advantage is thatlipid-containing receptors can be used as an integrated lipid in thecomplexes, that is, they can replace lipids that are used to build upthe complex (pat. lipid Iscom).

Among the receptor-binding components that are comprised by theinvention are, for example, bacterial toxins and their active bindingparts in the form of subunits or fragments or various modifications orderivatives of them, bacterial fimbriae or other adhesion molecules andtheir active binding parts and/or derivative structures. In many casesthese targeting structures are also relevant vaccine antigens, and thepresentation of such antigens on the surface of lipid-containingparticles, etc., for vaccination use are also part of the invention.

Iscom contains at least one glycoside, at least one lipid, and at leastone kind of antigen substance, particularly proteins and peptides. Thesecomplexes enhance the immunogenicity of the included antigens and mayalso contain one or more immunomodulatory (adjuvant-active) substancesand are described in EP 0 109 924 B1, EP 0 242 380 B1 and EP 0 180 546B1.

Matrix contains at least one glycoside, one adjuvant-active substanceand at least one lipid. Matrix has an immunoenhancing effect onco-administered antigenic substances, see EP 0 436 620 B1.

It has been shown that the lipids in these complexes can be partlyreplaced by lipid-containing receptors for antigen substances frommicroorganisms. In this way, the amount of antigen that binds to theparticle is appreciably increased.

In those cases where the complexes are iscoms, these iscoms are preparedas described in the European patent EP 0 109 942 B1. Here, virus,mycoplasma, bacteria, parasites, animal cells, containing antigens orantigenic determinants, especially proteins or peptides or isolatedexamples which have hydrophobic or amphiphatic regions, is mixed withone or more solubilizing agents, whereby complexes are formed betweenantigens or antigenic determinants and solubilizing agents, after whichthe antigens or determinants are separated from the solubilizing agentin the presence of, or are separated from the solubilizing agent anddirectly transferred to, a glycoside solution, containing cholesterol,phospholipid, and one or more glycosides (Quillaja components) withhydrophobic and hydrophilic domains in a concentration of at least thecritical micelle-binding concentration, whereby a protein complex isformed, which is then isolated and purified.

The lipids that are used are in particular those described in theapplicant's patent EP 0 109 952 B1, especially on page 3, and in EP 0436 620 B1, p. 7, lines 7-24. In particular, sterols such arecholesterol and phospholipids such as phosphatidyl-ethanolamine andphosphatidylcholine are used.

The lipids can also include lipophilic receptor molecules that bind tocell-binding components, especially antigens. Such receptors areglycolipids, for example the cholera toxin's receptor ganglioside GM1and fucosylated blood group antigen. The cell-binding components canthen function as transport molecules. They are bound to thelipid-containing receptor by a simple mixing with the complex thatcontains the receptor. Then the iscom or matrix molecule can be mixedwith the antigen that binds to the receptor.

It is possible to proceed from matrix that can be made by solubilizingat least one sterole in a solution agent, adding the glycoside or thesaponines and the other lipids, after which the solution agent may beremoved, if it unacceptable to the final product. Matrix is usuallytransferred to a water solution in which its separate parts are notsoluble. The solubilizing agent can be removed through eg gelfiltration, ultra filtration, dialysis, or electrophores. The matrix canthen be purified from surplus of sterole and saponine eg byultracentrifugation, through a density gradient or through gelfiltration. The solubilizing agent can be any of those mentioned in EP 0436 629 B1, p 5 row 24-45. The other components and the procedure arealso described in this document.

The glycosides that are used in the procedure can be those described inEP 0 109 942 B1 p 4 last paragraph. Especially saponines are used, suchas triterpensaponines, especially Quillaja saponins from Quillajasaponaria Molina or cell cultures from this tree or subcomponenetsthereof, especially those described in the applicant's European patentEP 0 436 620 B1 p 4 rows 19-46. These can be QHA, QHB, QHC, or othercompositions of Quillaja saponins. The glycosides are adjuvants andstructure-building elements in iscom and matrix. It is also possible toincorporate other adjuvants or immunomodulatory components thanglycosides in the iscoms or in the matrices as is mentioned in EP 0 436620 B1.

It is also possible to mix the transport molecule and/or the passengerantigen as a separate entity with an iscom particle in which thepassenger antigen or the transport (targeting) molecule has beenintegrated, or with iscom and/or matrix complex on which the passengerantigen or the transport molecule has been coupled, ie many combinationare possible. By definition, an iscom particle contains antigen andiscom-matrix lack antigen. Even other adjuvants or immunomodulatorycomponents can be mixed with the iscom and/or matrix complexes asseparate entities, ie they do not necessary have to be integrated in thecomplexes or coupled to these. Examples of such adjuvants are providedin Cox et. al., CRS, 1992. Usually, MDP, MTP, and avridin are used. Itis however advantageous to incorporate these adjuvants in iscom andmatrix when a lower dosage of adjuvants is required. It is also possibleto mix both the transport molecule and the passenger antigen withiscom-complex or matrix. In these cases, the iscom complex containsanother antigen molecule.

If the transport molecule(s) or passenger antigen(s) lacks hydrophobicor amphiphatic groups, they can be chemically coupled to the iscomparticle. Examples of such coupling procedure and coupling groups arefound in EP 0 242 380 B1 p 9 and in EP 0 426 620 B1 p 6 row 33-p 7 row6, where the coupling method is also described. They can be lipids as inexample 7 below.

The relative amounts of cholesterol, lipids and antigen that can be usedcan be found in the above-mentioned patents EP 0 109 942 B1, EP 0 180564 B1, EP 0 242 380 B1 and EP 0 436 620 B1.

The lipid-containing receptors can also be included in other lipidstructures such as liposomes, vesicles, micelles.

When the receptor is a hydrophobic protein such as a glycoprotein or afucosylated blood group antigen, it can be integrated in the lipidmolecule with a hydrophobic interaction. It can also be included iniscom as a protein share.

Besides the antigen that attach to the receptor, other antigens can bemade to attach to the receptor through the substitution of appropriategroups. Such appropriate groups, which can be attached to such otherantigens, can be parts of the antigen that attach to the receptor. Theseparts can be bound with familiar methods, for example those mentioned inEP 0 436 620 B1. It is also possible through gene-technologicalmanipulation to construct fusion proteins or peptides between an antigenand parts of it and the receptor-binding antigen. Other antigens thanthose that come from cholera and enterotoxic cholibacteria can therebybe bound to the GM1 receptor by substitution with parts of the CTB orLTB. Examples of this are given in Biochemia et biophysica Acta 1223(1994) 285-295 "Regeneration of active receptor recognition deomaisn onthe B subunit of cholera toxin by formation of hybrids from chemicallinactivated derivatives". Marc J. S. De Wolf. Wilfried S. H. Dierick. Inthis way, antigens that do not normally penetrate a mucous membrane canbe passed through the mucous membrane.

CTB is thereby a useful carrier for chemically and genetically producedantigens: Sanchez & Holmgren (Proc. Natl. Acad. Sci. USA, 86 481-485,1989) and Sanchez, Johansson et al. (Res. Microbiol. 141, 971-979,1990). With the aid of recombinant DNA techniques, foreign antigens havebeen bound to the antigen amino or carboxiy ends of the CTB subunit anda gene has been expressed that, via expression systems, has beendeveloped to produce the hybrid protein.

In the examples described to illustrate the invention, iscom matrix suchas lipid-containing particles and the cholera toxin B subunit (CTB) havebeen used as receptor-specific binding structures and as a relevantvaccine antigen.

The cholera toxin consists of an A subunit that exerts the toxinactivity and the B subunits that attaches the toxin to the plasmamembrane on the cell through a glycolipid (GM1). To reduce the toxicity,usually only the B subunit of the cholera toxin (CTB) is used as vaccineantigen to evoke immune response. The B subunit is not toxic and evokesa relatively weak immune response as compared to CT after local(mucosal) intranasal or systemic parenteral immunization, for examplesubcutaneous or intramuscular immunization, ie the B subunit has lowadjuvant activity when the activity refers to immunomodulating or immunoenhancing activity. CTB is also available as a recombinant DNA product(rCTB) (EP 368 819).

It is difficult to couple antigen covalently to CTB or LTB with highphysical and economic yields, since only a limited number of aminogroups and/or carboxy groups can be activated without seriously reducingthe antigenic activity of CTB and LTB or not to harm their capacity ascarrier molecules in the mucous membranes, and to target themselves andaccompanying antigens to lymphatic tissues and organs and cells to evokeimmune response. Even if a sufficient number of groups are available forchemical conjugation of antigen(s) on a carrier molecule, it is wellknown that it is difficult to receive good and economic yields from suchconstructions (Lovgren, et al., J. Immunol. Methods 173.237-243).

The utilization of transport proteins in iscoms has several advantages.This is especially evident for construction of oral, nasal, or rectalvaccines against infections. Such vaccines can contain a carrierconstruction with antigen and adjuvant component(s), supplemented forexample with CTB or LTB to localize the construction to lymphatic organsand cells in the intestinal tract and to target immune response tolymphatic tissues in other mucous membranes via GALT, MALT, NALT (Gut,Mucosal and Nasalfaryngial Associated Lymphatic Tissue, respectively),for example after administration in the respiratory tract or by directlocal mucosal application.

The iscom is larger than transport molecules like CTB or LT andtherefore there is room to conjugate or in some other way, for examplethrough hydrophobic or electrostatic binding, to incorporate chosenpassenger antigens and incorporate chosen adjuvant components.

In producing matrix, the weight ratio of sterol, second lipid, andglycoside is 0.2-10:0,2-10:1-100, preferably 1:1:5. If alipid-containing receptor is used, it can replace the other lipid(s)completely so that the ratio of sterol, lipid-containing receptor, andglycoside will be as above. It is also possible to use both thelipid-containing receptor and a second lipid, preferablyphosphatidylcholin or phosphatidyl ethanolamin and the receptor so thatthe ratio becomes sterol:second lipid:receptor:glycoside0.2-10:0.2-10:0.1-1:5-10, preferably 1:1:0.25:5. The amount of receptormolecule depends on the amount of target or antigen molecules one wishesto add on.

The constitutents can, in principle, be put in any ratio whatsoever. Ithas been proven that the finished product obtains the preferred weightratio between the included components and that the surplus does notenter. If a large amount of the second lipid, such as phospholipid, isused, the complex becomes fatty and fragile and easily falls apart. Toolittle of the second lipid makes it difficult for complex to be formed,and annularly ring-shaped subunits are formed. This can be determined byelectron microscopy.

Whether iscom or matrix has been formed can be confirmed by studying theproduct by electron microscopy. Typical matrix or iscom have acharacteristically open, spherical structure containing circularsubunits or parts of the spherical structure, as can be seen in FIG. 3in EP 0 109 942 B1. The iscoms have a lower sedimentation constant thanthe corresponding micelles and often a higher sedimentation constantthan the corresponding monomeric forms of protein or peptide. Matrix andiscom have a sedimentation constant of approximately 20 S.

The advantage of using lipid-containing receptors for bindingtarget-seeking or vaccine antigens is that it is possible to producematrix from glycoside, sterol, possibly a second lipid, and alipid-containing receptor and then simply mix the ready matrix with thetransport (targeting) molecule. The procedure is cheaper and simplerthan if one were to make ready-made iscom containing the ingredientsabove plus a transport antigen, or if one were to joint the antigen toready-made matrix using chemical conjugation methods.

When the antigen is integrated into iscom or is coupled chemically tomatrix, amino groups or carboxyl groups, which can constitute antigenicdeterminants, are modified. The antigenic determinants are denaturedwhen the antigen is activated for integration in iscom or when it iscoupled chemically to matrix or iscom (when two antigens are used, iscomalready contains at least one antigen). This means that the activeantigen amount is considerably reduced. Moreover, the recovery is lowcompared to when the antigen is allowed to bind to a lipid-containingreceptor. This can mean for example that in the preparation processapproximately five times more antigen is required as compared to when alipid-containing receptor is used. When a lipid-containing receptor isused, the process is considerably cheaper. Parallel to reducedincorporation, the amount of glycoside and adjuvant content per unit ofantigen is increased, which partly compensates the lower amount ofantigen as regards the achieved immune response, but at the same time,toxicity can increase because of the increased percentage of adjuvant.The immune response, on the other hand, becomes higher in principle whenusing receptor-binding of the antigen, while the original conformationalantigenic determinants are retained.

Another advantage presents itself when it is desirable to join atransport (targeting) molecule and a passenger antigen to iscom ormatrix. If iscom or matrix has been made with lipid-containingreceptors, there is more room for integrating passenger antigens in theiscom or for coupling it chemically to the matrix. By usinglipid-containing receptor, the binding of the passenger antigen is notinfluenced. It becomes easier to reach optimal conditions. Control ofthe amount of passenger antigen or transport (targeting) molecules,integrated in iscom or linked chemically to iscom or matrix, is madebetter. If iscom is made with both a transport (targeting) molecule andan antigen using the same methods, they may compete for the bindingregions and it is not possible to fully control the incorporations ofthe two antigens.

Especially in regard to the cholera antigen CTB, which has five bindingsubunit, it is possible to bind up to 13 times the weight amount of theGM1 receptor. There are still binding sites left in CTB, which can bindto cell receptors in the mucosa and serve as transport (targeting)molecules.

The weight ratio of sterol, second lipid, protein and glycoside is0.2-10:0.2-10:0.2-10:1-100, preferably 1:1:1:5-10 to be used withsubcutaneous administration. With oral or intranasal administration, theamount of glycoside can be higher in the ratio above, namely 1-200,preferably 5-20.

These amount apply both when first making matrix and then whenchemically coupling the antigens and when making iscom particles.

The procedure for preparing CTB (or LTB) iscoms entails mixing Quil A orQuil A components with a lipid mix containing cholesterol,phosphatidylcholin and Gal 1-3, Gal NAcb 1-4 (Neu Aca2-3), gal(GM1),which is a specific receptor for the cholera toxin (CT) and theheat-labile toxin from enterotoxic E. coli (LT) as well as theirsubunits CTB and LTB. Phosphatidylcholin (PC) can wholly or partly bereplaced with phosphatidylethanolamine (PE), whereby the amino group onPE constitutes a coupling group for antigen or other desired components.Unlike cholesterol, PC and PE are not essential to the iscom compositionbut can be replaced with other "soft" lipids.

Iscom or iscom matrix can be made in compositions containing asolubilizing agent such as water or physiological saline. Forsolubilizing agent, the composition can also include the detergent thatthe complex is made with if it is acceptable to human or veterinarymedicine. The compositions can also include other additives and fillersacceptable to human or veterinary medicine.

Such a composition can contain for example iscom complex and a fillersuch as physiological saline. It can also be composed of matrices mixedwith antigen. The vaccine can be made available in administrative formsthat contain an entity with matrix in a composition containing a fillerand a unit with the antigen in a composition containing a filler. Bothof these compositions are then intended to be administered on the sameoccasion.

The amount of iscom, matrix and antigen is chosen so that it will bepharmaceutically effective and can be decided by the expert. For humans,at least 1 μg, preferably 1-200 μg of the antigens, should be used,whereby economic opinion sets the upper limit. For animals, the dosagecan be at least 0.1 μg of the antigens, depending on the antigen and theindividual's size.

All cited publications and the Swedish priority application 9600648-1are incorporated herewith for reference.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1 shows that free rCTB (recombinant CTB) analyzed in a 10 to 50%sucrose gradient after ultracentrifugation is located at the top on thegradient, ie fraction 12-14. Cf FIGS. 2 and 3.

FIG. 2 shows iscoms with rCTB in a diagram where fractions in a 10-50%sucrose gradient after centrifugation are plotted against 1) theabsorbency at 595 nm for determination of the rCTB concentration usingthe method Bradford, and 2) CPM from analysis of ³ H cholesterol, aniscom-matrix component. Iscom matrix with rCTB and GM1 at the weightratio 13:1 is made according to example 1 and is then ultracentrifuged.Iscoms with rCTB are found in the fractions 6-9. Nearly all rCTB are tobe found in iscoms.

FIG. 3 shows a diagram of the same kind as FIG. 2. In making iscoms inthis trial, the rCTB concentration was 100 times greater than the GM1concentration w/w. Non-incorporated rCTB are to found in the upperBradford top, that is, higher up in the gradient in the fractions 10-12.

FIGS. 4A and 4B are bar graphs that show the ELISA titer in serum frommice after immunization with free rCTB and rCTB that has beenincorporated in iscom matrix.

The solid bars refer to subcutaneous immunization while the diagonalbars refer to intranasal immunization. FIG. 4A shows serum antibodytiters 5 weeks after the first immunization while FIG. 4B shows thetiter 6 weeks after the second immunization. The interval between theimmunizations was 6 weeks.

FIGS. 5A and 5B are bar graphs of the same kind as FIGS. 4A and 4B. Micewere immunized subcutaneously with 2 μg rCTB (solid bar) or intranasallywith 4 μg rCTB (diagonal bar). FIG. 5 shows serum antibody titers 5weeks after the first immunization while FIG. 5B shows the titer 6 weeksafter the second immunization. The interval between the immunizationswas 6 weeks.

FIG. 6 shows the antibody titers after different immunizations. rCTBmixed with matrix and rCTB bound in iscom evoke, after subcutaneousimmunization, serum antibodies of subclass. IgG2a and IgG1 unlike rCTB,which almost only evokes IgG1. FIG. 6A shows the result 14 days afterthe first booster immunization on day 42 while FIG. 6B shows the values4 days after the second booster immunization on day 110.

FIG. 7 shows the result after subcutaneous immunization of mice withrCTB bound to matrix, rCTB mixed with matrix, and only rCTB. The serumantibody response 14 days after the first immunization and 14 days afterthe second immunization are shown. The interval between theimmunizations was 42 days.

FIG. 8 shows the memory cell response after the second booster on day180 for the same immunization as in FIG. 6. Four days after the boosterdose, serum samples for antibody determination were taken.

The invention will now be described more closely regarding the followingprocedure examples.

EXAMPLE 1

Incorporating GM1 and rCTB in Iscoms

The cholera toxin (CT) is an effective adjuvant especially in localmucosal immunization. Even the cholera toxin's B subunit (CTB) isclassified as an adjuvant because of its target-seeking qualities inlocal immunization, but this activity is thus limited to a guidingfunction for the antigen to the lymphatic cells of the intestines. IfCTB is bound to iscom matrix or is incorporated in iscom, a formulationthat enhances the immune response is obtained, and is effective in localand parental immunizations. This effect is interesting in connectionwith vaccines against cholera and in choosing adjuvant for the otherantigens for local mucosal or parenteral immunizations. The recombinantcholera toxin, subunit B (rCTB) EP 0 368 819) is mixed with differentpreparations with:

MEGA-10(Bachem P1000 Decanoyl-n-methylglucamide), 20 weight-% in H₂ O;

Phosphatidylcholin (PC) (Sigma P 5763), 10 mg/ml dissolved in 20weight-% MEGA-10 in H₂ O.

Cholesterol (C) (Sigma C 8667), 10 mg/ml dissolved in 20 weight-%MEGA-10 in H₂ O;

GM1 (Sigma G7641), 10 mg/ml dissolved in 20 weight-% MEGA-10 in H₂ O;

Phosphatidylethanolamine (PE) (Sigma P2768), 10 mg/ml dissolved in 20weight-% MEGA-10 in H₂ O;

Quil A (Spikoside, Iscotec, Lulea), 100 mg/ml in H₂ O;

rCTB, 5 mg/ml in a buffer solution with 0.05 M TRIS (pH 7.5), 0.2 M NaCl0.0001 M Na2 EDTA, 0.003 M NaN3;

Phosphatidylcholin was mixed with cholesterol plus trace amounts ofradioactive cholesterol (³ H-cholesterol Amersham) in the proportion 1:1(100 mg av each lipid in 10 ml 20% MEGA-10) and with varying amounts ofGM1 from 1 μg to 7.5 μg (1 μg 1.7 μg, 2.5 μg, 4 μg, 5 μg, 7.5 μg) in 1.0ml PBS (phosphate-buffered physiological NaCl-solution), pH 7.2.

Into 1 ml of the six different variants ofphosphatidylcholin/cholesterol-GM1-solution, Quil A was added, to afinal concentration of 0.2%. The mixtures were sonicated in a Sonorex TK52 2×15 min. and were left at room temperature (RT) for 1 hour. Then themixtures were dialyzed against PBS, first for twenty-four hours in RTand then for twenty-four hours in a cold-room (+4° C.). That matrix wasformed could be seen by electron microscopy. Into each of the sixdifferent matrix variants, which differed regarding GM1 content, 100 μgrCTB were added. The mixtures were left for two hours in RT. The matrixparticles with associated rCTB, ie iscom, were purified bycentrifugation in a 10-50% sucrose gradient in PBS for 18 hours in a TST41.14-rotor (Kontron) at 39 000 rpm in 10° C. The gradient was collectedin 16 to 18 fractions. The fractions were analyzed in reference to rCTBusing the protein-determination method according to Bradford (Bradford,Analyt. Biochem., 72, 1976, 248-254) and was determinedcholorimetrically at 595 nm, and in reference to lipids throughdetection of ³ H-cholesterol, and electron microscopy to study thepresence of possible matrix or iscom structures. FIG. 1 shows freefractions 12-14. FIGS. 2 and 3 show the lipid (▪) and rCTB (□) amountsin the fractions when the ratio of rCTB:GM1 is 13:1 (FIG. 2), whereiscom with rCTB exists in the fractions 6-9, or 100:1 (FIG. 3), wherenon-incorporated rCTB lies higher up in the gradient, ie the fractions10-12.

Result

The greatest relative amount (weight) of rCTB that was completelyincorporated in the GM1 matrix, ie the iscom, was 13 times higher thanthe amount of GM1 (FIG. 2). In several other experiments, we have seenthe same ratio. If a higher amount of rCTB is added, the surplus rCTB isfound higher up on the gradient unassociated with ³ H cholesterol, whichshows that this rCTB is not incorporated. If a smaller amount of rCTB isadded, aggregates are formed through cross-linking because rCTB has fivepossible binding sites to GM1. Matrix with associated rCTB, ie iscom, isto be found in the fractions 6-9 (FIG. 2). Similar results are achievedwith phosphatidylethanol-amine in matrix or iscom instead ofphosphatidylcholin (results not presented).

Conclusion

rCTB can effectively be bound to matrix that contains the glycolipidGM1. An addition of an appropriate amount of GM1 during the matrixpreparation implies an efficient procedure method.

EXAMPLE 2

In this example, it is shown that rCTB incorporated in iscom evokes ahigher antibody response than free rCTB.

GM1 matrix was prepared in the same way as in example 1.Phosphatidylcholin/cholesterol and GM1 (PC/C/GM1 and Quil A) in theproportion of 1:1:0.25:5 was mixed with MEGA-10 (final concentration:2%). The mixture was dialyzed in the same way as in example 1. rCTB wasadded in an amount (weight) that was 13 times higher than the amount ofGM1. In the same way as in example 1, formed complex was analyzed withEM and sucrose gradient centrifugation. Gradient fractions were analyzedas in example 1 regarding cholesterol and protein (rCTB). Iscoms withincorporated rCTB were thereafter saved for use in immunizationexperiments.

Six groups of eight mice each were immunized subcutaneously with 2 grCTB or with 4 g rCTB intranasally on two occasions within a six weekinterval (see FIGS. 4A and 4B). rCTB was present either in a free form,ie mixed with matrix without GM1, or bounded to matrix via GM1. Twovariants of GM1 matrix according to the above were used in the weightproportion 13:1 or 25:1 (rCTB:GM1 depending on weight), ie saturated oroverly saturated in regards to the proportion rCTB/GM1.

Group A Free rCTB, 2 μg rCTB inj. s. c. 0 μg Quil A

Group B Free rCTB, 4 μg rCTB inj. i. n. 0 μg Quil A

Group C Iscom; 2 μg rCTB (13×GM1) inj. s. c. 3 μg Quil A

Group D Iscom; 4 μg rCTB (13×GM1) inj. i. n. 6.1 μg Quil A

Group E Iscom; 2 μg rCTB (25×GM1) inj. i. n. 1.6 μg Quil A

Group F Iscom; 4 μg rCTB (25×GM1) inj. i. n. 3.2 μg Quil A

The antibody titers in serum were measured using ELISA at differenttimes according to FIGS. 4A and 4B.

In the ELISA test, the ELISA plates (Nunc, Roskilde, Denmark) wereincubated with a 50 mM carbonate buffer, pH 9.5, containing 2 μgrCTB/ml. Serum samples from the mice were diluted in series. The ELISAplates were treated with the diluted serum solutions. Bound miceantibodies were detected with peroxidase-conjugated rabbit-anti-mouseconjugate (Dakopatts) and as a substrate. TMB, H₂ O₂ (EC diagnostics,Uppsala) was used.

Result

The results are outlined in FIGS. 4A and 4B, which show serum antibodytiters measured in ELISA 5 weeks after the first subcutaneous andintranasal immunizations with rCTB (A) and 6 weeks after the secondimmunization (B). The interval between immunizations was 6 weeks. rCTBincorporated in iscom in the proportion of 13:1 (rCTB:GM1(weight))evoked, after two subcutaneous immunizations with 2 μg rCTB, a titer of87 000. Iscoms with a rCTB:GM1 ratio of 25:1 evoked titers of 50 000.Corresponding serum antibody titers for two subcutaneous immunizationswith free rCTB were 8 600.

After two intranasal immunizations with rCTB in iscom (13:1) (rCTB:GM1)serum titers of 21 000 were obtained, while 25:1 (rCTB:GM1) evoked serumantibody titers of 33 000. Free rCTB evoked, after two intranasalimmunizations. ELISA titers in serum of 19 000.

Conclusion

rCTB in iscom is, after subcutaneous immunization, more immunogenic thanfree rCTB, but there was no significant difference after intranasalimmunization.

EXAMPLE 3

In this experiment, matrix was used as adjuvant with non-incorporatedantigen in subcutaneous and intranasal immunization.

Matrix without GM1 was prepared basically as described in example 1 withthe only difference that GM1 was excluded. The weight proportions ofphosphatidylcholin/cholesterol/Quillaja were 1:1:5. Lipids weredissolved in 20% MEGA. Dialysis was conducted as in example 1. Matrixwas analyzed and characterized as in example 1 using EM and analyticalsucrose gradient centrifugation. Matrix with GM1 was prepared as inexample 1 and rCTB was incorporated in the proportion 13:1 (rCTB:GM1).When GM1 was excluded from matrix, no biding of rCTB to matrix occurred.

Eight mice per group were immunized subcutaneously with 2 μg rCTB iniscoms or mixed with matrix as adjuvant or intranasally with 4 μg rCTBin iscoms or mixed with matrix as adjuvant. Two immunizations werecarried out within a six week interval.

Group C: Iscom: 2 μg rCTB subcutaneously, 3 μg Quil A

Group D: Iscom: 4 μg rCTB intranasally, 6.1 μg Quil A

Group G: Matrix mixed with: 2 μg rCTB subcutaneously, 3.0 μg Quil A

Group H: Matrix mixed with: 4 μg rCTB intranasally, 6.1 μg Quil A

The antibody titers in serum were measured using ELISA and the titersare given as the dilution that gives the absorbence of 1.0.

Results

The results are summarized in FIG. 5, which shows serum antibodyresponse after immunization with 2 μg rCTB administered s. c. or 4 μgrCTB administered i. n. measured in ELISA 5 weeks after the firstimmunization (A) and 6 weeks after the second immunization (B). Theinterval between immunizations was 6 weeks. After two subcutaneousimmunizations with rCTB mixed with matrix as adjuvant, an average titerof 91,000 was induced, compared to 87,000 for rCTB in iscom form. Aftertwo intranasal immunizations, 54,000 was induced for rCTB mixed withmatrix, compared to 21,000 for rCTB in iscom.

Conclusion

After subcutaneous immunization, the serum antibody titers induced byrCTB in iscom form were almost as high as that induced by rCTB mixedwith matrix as adjuvant. After intranasal immunization, twice as hightiters after immunization with rCTB mixed with matrix were induced aswith rCTB in iscom form. It is interesting to note that matrix in freeform has as strong an adjuvant effect on antibody response as the iscomform of rCTB. In the matrix formula, twice as much Quillaja wasincluded.

Above all, it is surprising that matrix in free form has an adjuvanteffect after local mucosal administration with rCTB, which in itself hasan adjuvant effect in the form of targeting when immunized throughmucous membranes.

EXAMPLE 4

One of the tasks for an adjuvant is to evoke a strong immune responsethat can be measured as an antibody response or as a cell-mediatedimmune response. Another of its tasks is to evoke the desired type ofimmune response, which eg can be read in IgG subclasses that reflectT-helper cell response identified with cytokine production. In thisexperiment, it is shown that rCTB in free form without adjuvant evoke aserum antibody response that is focussed to subclass IgG. By mixing rCTBwith matrix or by incorporating rCTB in iscom, serum antibodies are alsoevoked against rCTB in the subclass IgG2a, which is associated with aTH1 response.

Eight mice per group (three groups) were immunized twice subcutaneouslyat a 6-week interval with 2 μg rCTB without adjuvant or with 2 μg rCTBmixed with matrix or with 2 μg rCTB-iscom.

The serum antibody responses were measured using ELISA according to atime schedule that can be seen in FIG. 6. The distribution of serumantibodies in classes and subclasses was analyzed using ELISA by use ofclass and subclass-specific antisera (Dakoparts, Denmark).

Results

Free rCTB without adjuvant mainly evoked an IgG1 response against rCTBwhile no antibodies in subclass IgG2a could be found. Both rCTB-iscomand rCTB mixed with matrix evoked both IgG1 and IgG2a antibodies againstrCTB (FIG. 6A) 14 days after the first booster immunization, day 42.Even after a second booster on day 110, free rCTB without adjuvant didnot evoke an IgG2 response, while iscom and rCTB mixed with matrix gavea clear IgG2 response (FIG. 6B) 4 days after the 2nd boosterimmunization.

Conclusion

There are differences in quality regarding the serum antibody responsein rCTB in free form without adjuvant as compared to rCTB provided withmatrix as adjuvant or bound to iscom. Both matrix and iscom with rCTBevoke antibodies against rCTB of subclass IgG2a as well as IgG1, unlikefree rCTB, which is only able to evoke IgG1-antibodies.

EXAMPLE 5

The effect of vaccines against infections depends not only on the directeffect that the evoked immune response has, but also on the inducedmemory cells that are recruited in connection with infections. Thememory cell function is especially important a long time after thevaccination, when the evoked immunity will have become low. A strongmemory cell response, that can be recruited quickly at the time ofinfection, is therefore desirable.

Eight mice per group (3 groups) were immunized twice subcutaneously atan 8-week interval with 2 μg rCTB without adjuvant, with rCTBincorporated in iscom, or rCTB mixed with matrix. The antibody responsewas measured using ELISA, each group was divided into two subgroups of 4mice each. A second booster immunization was executed on day 180 andblood tests for serum were taken 4 days later.

Results

The results can be seen in FIG. 7. After the first immunization (day14), the highest immune responses were evoked by rCTB mixed with matrix(17,000) and rCTB iscom (9,000) rCTB without adjuvant induced titers ofapproximately 1,000. Two weeks after the second immunization (theinterval between immunizations was 42 days), mice in all the groups hadincreased their serum antibody titers appreciably. The highest titerswere found in the matrix group (approximately 57,000) and the iscomgroup (35,000), while the titers for the group that was vaccinated withrCTB without adjuvant had titers of approximately 6,000.

After the third immunization (i.e. the second booster) on day 180, i.e.140 days after the second immunization, the mice that were immunizedwith free rCTB had antibody titers of approximately 8,000, i.e. justabout the same titers as after the second immunization. Mice that wereimmunized with iscom or with rCTB mixed with matrix responded after the3rd immunization on day 180 with increased serum antibody titers formatrix (approx. 90,000) and iscom (approx. 70,000) (FIG. 8). Serum wastaken 4 days after the 3rd immunization for antibody tests.

Conclusion

The strong antibody increase in serum in mice that were reimmunized along time after the earlier immunization (140 days) with rCTB iscom orwith rCTB mixed with matrix shows that a strong memory response has beenevoked by the previous immunizations. rCTB without adjuvant, however,did not show any immune response that can be boostered after a longtime.

EXAMPLE 6

rCTB was preincubated at 20° C. for 1 hour with GM1-containing matrix inproportions that were tested in advance so that preparation (A) shouldsaturate matrix with rCTB, and so that preparation (B) would give ansupersaturation so that approx. half the amount of rCTB could not bindto matrix. After incubation, the matrix was purified from unbound rCTBthrough centrifugation.

Preparations A and B were then used for peroral, intranasal, orintraperitoneal immunization of 8-10-week-old C57/B1 mice. Groups of 3mice in each group were given 3 doses with a 2-week interval between thedoses. Each dose contained 17 μg rCTB and 26 μg Quillaja for peroralimmunization, half the dose for intranasal, and a sixth forintraperitoneal immunization.

Three more groups of mice were given peroral, intranasal, andintraperitoneal immunizations, respectively, with corresponding amountsof matrix (not containing GM1) mixed with rCTB.

One week after the third dose, the animals were killed, exsanguinated,and perfused with PBS-heparin whereafter lung tissue was taken from bothlungs and crushed, and pieces of the intestinal canal were taken fromthe upper, middle and lower parts of the small intestine and werecrushed. The tissue was frozen at -30° C. and was then thawed andsuspended in PBS-1% saponine (1 ml per 1 mg tissue) and was extracted incold (+4° to 10° C.) over the night. The tissue extract and sera werethen titered for specific antibodies against CTB using GM1-ELISA.

The results can be seen in Table 1. It is evident that all of thepreparations A-C give high serum antibody responses afterintraperitoneal and intranasal immunization and that intranasalimmunization with (A) and (B) stimulate good local IgA antibody titersin tissue extract from respiratory tract cells.

                  TABLE 1                                                         ______________________________________                                        ANTIBODY TITERS IN MICE IN SERUM AND TISSUE EXTRACT                           AFTER INTRAPERITONEAL (LP): INTRANASAL (IN) OR ORAL                           IMMUNIZATION WITH B-SUBUNIT (rCTB) FROM THE CHOLERA                           TOXIN IN ISCOM                                                                       LUNG      INTESTINE  SERUM                                                    IgA   IgG     IgA     IgG  IgA   IgG                                   ______________________________________                                        A      PO    <2      15    42    <2   35    1400                                     IN    2900    20,700                                                                              21    140  970   >400,000                                 IP    <2      7000  18    350  <2    260,000                           B      PO    <2      14    22    20   10    6,800                                    IN    1550    7000  240   510  1420  >400,000                                 IP    <2      220   100   100  10    120,000                           C      PO    <2      76    39    39   <2    99,000                                   IN    340     5,400 28    415  350   380,000                                  IP    --      6,700 --    450  --    >400,000                          D      PO    0       --    30    N.d. --    --                                =CTB   IN    500     --    50    N.d. --    30,000                            E      PO    20      2,100 4,300 800  1,300 87,000                            = CTB +                                                                              IN    10,000  46,000                                                                              500   5,100                                                                              40,000                                                                              600,000                           CT     IP    N.d.    2,300 100   480  630   79,000                            ______________________________________                                         3 immunizations, 3 mice per group. Median titers are shown.                   Mice were immumzed 3 x                                                        A = PC/C + GM1 + rCTB 13 x (saturated)                                        B = PC/C + GM1 + rCTB 25x (supersaturated)                                    C = PC/C - GM1 + rCTB (PC/C and rCTB separate)                                D = rCTB                                                                      E = rCTB + choleratoxin                                                       Dilution expressed as 1/x is < 5                                              PO = peroral administration 20 μl (17 μg CTB & QA 26 μg)             IP = intraperitoneal 3 μl                                                  IN = intranasal 10 μl                                                 

EXAMPLE 7

This example shows that an iscom containing matrix with GM1 and to whichrCTB has been bound and incorporated together with an Ovalbumin (OVA)which has been provided with a lipid tail evokes antibody responseagainst rCTB and against OVA after one intranasal (IN) immunization.

Lipidating OVA

Reagent

1 mg OVA

1 mg phosphatidylethanolamin (PE), with small amounts of ¹⁴ C-labelledPE

1.4 mg N-hydroxisulfosuccinimide

38.4 mg 1-ethyl-3-(3-dimethylaminopropyl)-charbodiimid-HCl

H₂ O to a volume of 2 ml.

The mixture was incubated for 2 hours on a shakeboard in roomtemperature.

This OVA was incorporated in iscoms with and without GM1, analogouslywith the method described earlier in example 1. The iscoms werecharacterized through electron microscopy (EM) and analytic sucrosegradient centrifugation in the same was as in example 1.

In iscom preparation the following ingredients were used:

    ______________________________________                                        Lipidated OVA, 2 ml                                                                               300 μg                                                 Cholesterol        1000 μg                                                 Spikoside (Quil A) 5000 μg                                                 H.sub.2 O to a volume of 2.15 ml                                              ______________________________________                                    

For preparation, see example 1.

When the iscoms were also to contain GM1, the following ingredients wereused:

    ______________________________________                                        Lipidated OVA       300 μg                                                 Cholesterol        1000 μg                                                 GM1                 50 μg                                                  Spikoside (Quil A) 5000 μg                                                 Total volume 2.2 ml (H.sub.2 O).                                              ______________________________________                                    

In preparing OVA-rCTB iscoms, OVA-GM1 iscoms were mixed with 13 timeshigher amounts (weight) rCTB than GM1. The amounts were calculated inthe same way as in EP 180 562, example 2.1.

Five groups of eight mice each were immunized intranasally twice with asix-week interval according to the following schedule:

    ______________________________________                                        Group A                                                                              OVA (free), 10 μg intranasally                                      Group B                                                                              OVA-iscom, 10 μg antigen intranasally                               Group C                                                                              OVA-iscom + rCTB (free), 10 μg of each antigen intranasally         Group D                                                                              OVA-rCTB iscom, 10 μg of each antigen intranasally                  Group E                                                                              OVA-rCTB iscom, 2 μg of each antigen subcutaneously                 Group F                                                                              OVA free + rCTB free 10 μg of each antigen intranasally             ______________________________________                                    

3 weeks after the first immunization and 2 weeks after the secondimmunization, serum samples were collected. Lungs were prepared 2 weeksafter the first and second immunizations for extraction of IgAantibodies. Antibody titers in serum and lung extract were determined inthe same way as in examples 1 and 2.

Results After One Immunization

The mice in group D responded with significant levels of antibody titersagainst rCTB in serum. Significant antibody titers against rCTB weredetected in lung extract. Lower antibody titers were obtained againstOVA in both serum and lung extract.

After subcutaneous immunization (group E) approx. the same serumantibody levels against CTB as against OVA were obtained. No antibodyresponse was measured against rCTB or OVA in lung extract.

In the mice in group C, that had been immunized intranasally withOVA-iscoms plus free rCTB, antibody titers against rCTB were measuredwith ELISA serum antibody titres that were of the same level as those inthe mice in group D. No antibody titers against OVA could be measured inserum. In lung extract, significant titers against rCTB were measured,but no or very little antibody response could be measured against OVA.

In the mice in group F moderate antibody titers were obtained in serumand lung extract against rCTB after primary immunization (i. n.) but notagainst OVA.

In mice in the remaining groups (A, B) no antibody titers against OVAcould be measured in serum nor in lung extract.

Results After the Second Immunization

High antibody titers against OVA in serum were measured in the mice ingroups D and E, ie the mice that were immunized intranasally orsubcutaneously, respectively, with OVA-rCTB iscoms.

In lung extract from the mice in group D, antibody titers against bothrCTB and OVA were measured.

No or very low titers were measured in lung extract in mice that wereimmunized subcutaneously with OVA-rCTB iscoms (group E).

In the mice in group C, high antibody responses against rCTB in serumwere measured, but very low serum titers were obtained against OVA. Inlung extract antibody titers against rCTB but not against OVA weremeasured.

In the mice in group B (OVA-iscom) low titers against OVA in both serumand lung extract were measured. Free OVA (group A) evoked no detectableantibody titers, in either serum or lung extract.

After the second immunization several-fold serum antibody increase oftitres against rCTB but not against OVA was obtained. After the secondimmunization, IgA antibody titers against rCTB were measured in the lungthat were significant but no antibody response was measured against OVA.

Conclusion

The results show that iscoms containing rCTB as transport (targeting)molecules and OVA as passenger antigen effectively induce antibodyresponse against both rCTB and OVA in lung extract and serum. Only OVA,OVA iscoms (iscoms with only passenger antigen) or free OVA plus rCTBiscoms evoked no or very low antibody response against OVA in serum andlung extract.

We claim:
 1. Lipid-containing particles, chosen from iscoms andiscom-matrix, comprising at least one receptor for antigen substancesfrom microorganisms, bacteria toxins, fimbria, adhesins and bindingactive parts thereof, which receptor has been integrated in theparticle, and is chosen from lipid-containing receptors or receptorsthat are hydrophobic.
 2. Lipid-containing particle according to claim 1composed of iscoms containing at least one glycoside, at least one lipidand at least one hydrophobic protein or peptide-containing antigen,further comprising a lipid-containing receptor.
 3. Lipid-containingparticle according to claim 1 composed of iscom-matrix containing atleast one glycoside and at least one lipid, further comprising alipid-containing receptor.
 4. Lipid-containing particle according toclaim 3, containing at least two antigens or one antigen and onetarget-seeking molecule, wherein one antigen is bound to areceptor-binding molecule.
 5. Lipid-containing particles according toclaim 1, wherein the receptor molecule is a lipid-containing receptor.6. Lipid-containing particles according to claim 1, wherein the receptormolecule is the lipid-containing receptor GM1 of the cholera toxin andthe antigen is the cholera toxin or a subunit thereof, orimmuno-logically closely related proteins including heat-labileenterotoxin from E. coli, or its subunit that binds to the receptor. 7.Lipid-containing particles according to claim 1, wherein the receptormolecule is a hydrophobic protein.
 8. Lipid-containing particlesaccording to claim 7, wherein the receptor molecule is a glycoprotein orfucosylated blood group antigen.
 9. Lipid-containing particles accordingto claim 7, wherein the receptor molecule is the glycoprotein that bindsto head-labile enterotoxin from E. coli or a subunit thereof and whereinthe antigen is the enterotoxin from E. coli or a subunit thereof. 10.Vaccine preparation for prophylaxis or immunotherapy, containingparticles according to claim 1.